Rotavirus gastroenteritis causes more than 500 000 deaths every year in young children worldwide. Rotaviruses (RV), members of the family of Reoviridae, is the single most important causative agent of severe diarrhea in young children also in developed countries, resulting in fewer deaths than in developing countries but numerous hospitalizations at high cost. Live oral rotavirus vaccines have been available since 2006. Both the WHO and many indivictual countries now recommend vaccination of all healthy children against rotavirus.
In the development of a live oral rotavirus vaccine, Professor Timo Vesikari of the University of Tampere has played a key role. The first clinical trials of any rotavirus vaccine were conducted in Tampere in 1982. Prof. Vesikari and his team were instrumental in the pivotal trials establishing the efficacy and safety of the two currently licensed live oral rotavirus vaccines, the bovine-human reassortant pentavalent vaccine RotaTeq® (Merck) and the human rotavirus vaccine Rotarix® (GSK) (Vesikari et al. Safety and efficacy of a pentavalent human-bovine (WC3) reassortant rotavirus vaccine. N Engl J Med 2006;354:23-33; Vesikari et al. Efficacy of human rotavirus vaccine against rotavirus gastroenteritis during the first 2 years of life in European infants: randomised, double-blind controlled study. Lancet 2007;370:1757-63).
The currently available live attenuated oral rotavirus vaccines, while efficacious and successfully implemented in many countries, have potential safety issues that may limit their use in the long run. An earlier live oral rotavirus vaccine, based on rhesus rotavirus (RotaShield®, Wyeth), was withdrawn in the USA in 1999 because of association with intestinal intussusception, which may have occurred in about 1 in 10 000 recipients of the first dose of the vaccine. The currently licensed rotavirus vaccine does not involve such a great risk but a rare association cannot be excluded.
Furthermore, in 2010, it was discovered that both licensed live rotavirus vaccines contained porcine circovirus (PCV) DNA. Although the significance of this finding is unknown, it caused temporary suspension of one of the vaccines and a decrease in rotavirus vaccination overall. Both of these issues are inherent to live vaccines only, and together emphasize the need to develop non-live alternatives for rotavirus vaccines.
A rotavirus genome consists of 11 segments of double stranded RNA held in the inner core of the three-layered virus. The three layers consist of a core protein VP2 bound to dsRNA, an inner capsid protein VP6, and an outer capsid glycoprotein VP7 with hemagglutinin spike protein VP4. The major capsid protein VP6 determines viral group specificity and is the most conserved, immunogenic, and abundant rotavirus protein. The outer capsid proteins VP7 and VP4 contain neutralizing epitopes and induce protective immunity on the basis of neutralizing antibodies.
The mechanism of active protection induced by live oral rotavirus vaccines is not fully known. Surface proteins VP7 and VP4 are known to induce serotype specific neutralizing antibodies. However, there is significant cross-protection between serotypes that cannot be explained by serotype-specific immunity. VP6 is an immunodominant protein in rotavirus infection and after vaccination. Although VP6 does not induce neutralizing antibodies it induces heterologous rotavirus specific immunity.
The first rotavirus recombinant VP6 (rVP6) protein was produced from the rBV expression system more than two decades ago (Estes M et al. 1987). VP6 alone forms oligomeric structures including tubules, spheres and sheets in vitro composed of a variable number of trimers (Lepaualt J, Embo J, 20, 2001). Co-expression of VP2 and VP6 in rBVs results in the formation of double-layered virus-like particles (dl VLPs). Coexpression of VP2, VP6, and VP7 (with or without VP4) leads to triple-layered VLPs resembling native infectious rotavirus particles. A majority of the immunogenicity and vaccine efficacy studies in animal models has accomplished using different rotavirus VLPs or non-human recombinant VP6 protein with an adjuvant. No human clinical trials with the non-live subunit rotavirus protein vaccines using either VLPs or the recombinant VP6 protein have been accomplished so far.
After elimination or reduction of rotavirus in many areas, the relative role of norovirus as a causative agent is increasing. Noroviruses (NV) are members of the family Caliciviridae causing sporadic acute nonbacterial gastroenteritis in humans of all age groups, and are associated with outbreaks of gastroenteritis worldwide. NV cause annually approximately 1 million hospitalizations and more than 200 000 deaths worldwide in children less than 5 years of age. After rotavirus, the second most important viral cause of acute gastroenteritis in young children is norovirus.
A norovirus genome consists of a single stranded RNA of about 7.6 kb that is organized into three open reading frames (ORF 1-3). The ORF1 codes for RNA-dependent RNA polymerase similarly to other ssRNA viruses; the ORF2 encodes the major capsid protein VP1 and the ORF3 codes a small structural protein VP2. Most NVs affecting humans belong to two genogroups (GI and GII), and these two genogroups are divided into at least 8 GI and 17 GII genotypes. In recent years, it is the genotype GII-4 that has been primarily responsible for the majority of sporadic gastroenteritis cases and outbreaks.
A unique feature of the capsid VP1 protein is its ability to self-assemble into the empty virus-like particles (VLPs). Cloning of genogroup I Norwalk virus capsid gene into a recombinant baculovirus (rBV) has led to the production of the first norovirus VLPs twenty years ago (Jiang et al. 1992). These VLPs are morphologically and antigenically similar to the native NV. The three-dimensional structure of norovirus VLPs, viewed by using electron cryomicroscopy and computer image processing techniques, shows that the norovirus capsid forms a T=3 icosahedral symmetrical structure containing 180 molecules of the VP1 capsid proteins organized into 90 dimers with a diameter of 38 nm. Norovirus VLPs are widely used as a source of antigen in diagnostic serological assays, as well as for development of candidate vaccines against noroviruses. Although the receptor/s for norovirus binding and entry is/are not completely elucidated, it has recently been found that NV recognize human histo-blood group antigens (HBGAs) as receptors. Among the HBGAs, the most commonly encountered blood groups are ABO (ABH) and Lewis. These complex carbohydrates are found on the red blood cells and mucosal epithelial cells or as free antigens in biological fluids. Further, it has recently been found that the recognition of HBGAs by NV is strain-specific, and several distinct receptor binding patterns have been identified.
For norovirus, a live vaccine is not an option, because noroviruses cannot be cultivated in a cell culture. Therefore, the candidate vaccines for norovirus have been and are likely to be either VLP vaccines or soluble antigen vaccines.
The use of non-replicating subunit vaccines and subviral particles in modern vaccine design started in the mid 80's with the discovery of Hepatitis B surface antigen (HBsAg) particles found in blood of HB infected patients. These vaccines are generally safe as they are deprived of any live attenuated or inactivated viruses or their genetic material, and they are relatively easily and cost effectively produced in high quantities. An example of a subunit protein vaccine are virus like particles (VLPs) which mimic empty shells of live viruses and therefore possess antigenic and immunogenic properties similar to those of the live virus. Vaccine induced serum neutralizing antibodies are important to for protection against viral infections. Essential features of VLPs include that they resemble the natural virus and therefore retain neutralizing epitopes which are conformation-dependent.
There are several intriguing features or major attributes of NV VLPs and RV VP6 protein which make them promising vaccine candidates. Because of their repetitive, multivalent structures, VLPs are extremely immunogenic. The presentation of an antigen in a highly organized, dense, repetitive array on the surface of VLPs provokes strong antibody responses at very low doses, whereas the same antigen presented as a monomer is normally nonimmunogenic. B cells are efficiently activated by these repetitive structures (as are VLPs or rVP6 trimers organized into hexagons and packed into the higher order structures, e.g. tubules) which lead to cross-linking of B cell receptors on the cell surface. The particulate nature of VLPs, especially in a size range of around 40 nm, which is optimal for uptake of nanoparticles by professional antigen presenting cells (APC), namely dendritic cells (DC), via macropinocytosis and endocytosis (Fifis T., J Immunol, 2004, 173). Therefore, VLPs similarly to live viruses directly activate and mature DC without the need for other cells. DC play a central role in activating innate and adaptive immune responses and are involved in long lived memory IgG production and are the only APC capable of activating naïve T cells. VLPs efficiently prime CTL in the absence of intracellular replication (Keller S A et al., Intro, J Immunol, 2010). Therefore, VLPs are efficient in stimulating both cell mediated immunity (CMI) and humoral immune response.
As already mentioned above, VP6 is the most abundant and immunogenic subgroup-specific antigen of rotavirus. The ability of VP6 to form multimeric structures and the strong immune response that VP6 can elicit make it an excellent rotavirus vaccine candidate. VP6 does not induce neutralization antibodies to rotavirus but instead induces heterotypic cross-protective immunity by eliciting strong T helper (Th) cell responses which promote cross-reactive immunity (Burns J W et al., 1996 Science; Parez N et al, 2004, J Virol). VP6-specific CD4+ Th cells provide cognate help to B cells specific for neutralizing epitopes on the VP7 and/or VP4 molecules (Esquirel F R, Arch. Virol 2000, 145, 813). In addition, VP6-specific CD4+ Th cells were shown to protect against murine rotavirus infection either by a direct cytotoxic mechanism in mucosa or by antiviral cytokines production.
Given the severity of rotavirus and norovirus infections and deficiencies in the currently available vaccines, there is a need for both non-live norovirus and rotavirus vaccines, especially for the prevention of acute gastroenteritis in childhood. Immune responses to NV and RV are complex, and the correlates of protection are not completely elucidated. Collectively, the above-described unique properties attributed to the VLPs including NV VLPs and to the VP6 protein of RV suggest that a vaccine consisting of these two components represents a viable strategy to immunize against NV and RV infection.